The endoplasmic reticulum (ER) is an organelle specialized for protein folding and assembly of membrane proteins and of proteins destined for trafficking tolysosomes and the extracellular space. Newly synthesized lysosomal, secretory, and membrane proteins are translocated into the lumen of the ER that provides an oxidizing environment and contains a multitude of ER resident proteins that facilitate the folding process (reviewed by Gething and Sambrook, Nature 355:34-44 (1992); Hartl, F. U., Nature 381:571-579 (1996)). The transcription of many of the genes encoding ER resident proteins, such as BiP (immunoglobulin binding protein or GRP78), is upregulated in response to glucose deprivation (Lee, A. S., Trends Biochem. Sci. 12:20-30 (1987)), in response to conditions that disrupt protein folding in the ER, and in response to the presence of unfolded or unassembled proteins in the ER. Lee, A. S., Trends Biochem. Sci. 12:20-30 (1987); Kozutsumi, Y. et al., Nature 332:462-464 (1988); Dorner, A. J. et al., J. Biol. Chem. 264:20602-20607 (1989). Thus, an unfolded protein response (UPR) exists in cells that detects unfolded protein in the ER lumen to transduce a signal(s) across the ER membrane to activate transcription of selective genes in the nucleus. Kozutsumi, Y. et al., Nature 332:462-464 (1988).
Although little is known about the mechanism of the UPR signal transduction pathway in higher eukaryotes, studies from the budding yeast, Saccharomyces cerevisiae, demonstrate the existence of a complex unique signaling pathway between these two organelles. Mori, K. et al., EMBO J. 11:2583-2593 (1992). Characterization of the promoters of the genes encoding ER resident proteins e.g. KAR2 (yeast BiP), demonstrated that they share a highly conserved cis-acting regulatory Unfolded Protein Responsive Element (UPRE), that is necessary and sufficient to mediate the response to unfolded protein in the ER. Mori, K. et al., Cell 74:743-756 (1993); Cox, J. S. et al., Cell 73:1197-1206 (1993). By using genetic approaches, Ire1p/Ern1p, an ER type 1 transmembrane protein that contains a Ser/Thr protein kinase domain in its carboxy terminus, was identified as the UPR proximal sensor that monitors the status of unfolded protein inside the ER lumen. Cox, J. S. et al., Cell 73:1197-1206 (1993); Mori, K. et al., Cell 74:743-756 (1993). Ire1p was originally identified as a gene required for inositol prototrophy in S. cerevisiae. Nikawa, J. et al., Mol. Microbiol. 6:1441-1446 (1992). The kinase activity of Ire1p is essential to transmit the UPR signal from the ER to induce specific gene transcription in the nucleus. Mori, K. et al., Cell 74:743-756 (1993); Shamu, C. E. et al., EMBO J. 15:3028-3039 (1996). Cox and Walter (J. Biol. Chem. 264:20602-20607 (1996)) subsequently reported that Ire1p directly regulated biosynthesis of Hac1p, a transcription factor that binds specifically to the UPRE. Recent studies demonstrate that HAC1 mRNA is synthesized as a precursor that is inefficiently translated. Upon activation of the UPR, Ire1p elicits an endonuclease activity that specifically cleaves an intron from HAC1 mRNA. Subsequently, the tRNA ligase Rlg1p is required to splice together the 5′ and 3′ cleaved fragments to yield a product that is efficiently translated. Cox, J. S. et al., J Biol. Chem. 264:20602-20607 (1996); Sidrauski, K. et al., Cell 90:1031-1039 (1997); Kawahara, T. et al., Mol. Biol. Cell 8:1845-1862 (1997); Chapman, R. E. et al., Curr. Biol. 7:850-859 (1997)). The increased level of Hac1p leads to the transcriptional activation of genes containing a UPRE.
While the molecular mechanisms signaling the yeast UPR are well characterized, the mechanisms signaling the UPR in mammalian cells remain elusive. A conserved promoter region, the glucose-regulated core sequence, in several mammalian genes encoding for ER proteins was identified as a potential cis-acting regulatory element equivalent to the yeast.UPRE. Resendez, E. J. et al., Mol. Cell. Biol. 8:4579-4584 (1988). Despite the sequence similarity between the mammalian glucose-regulated core sequence and the S. cerevisiae UPRE, no single element in this promoter region appears necessary and sufficient to mediate transcriptional induction as described for the UPRE in yeast cells. In addition, although transcriptional activation in response to conditions that disrupt protein folding in the ER correlates with changes in activities of protein kinases and phosphatases (Resendez. E. et al., J. Cell Biol. 103:2145-2152 (1986); Koong et al. 1994; Cao, X. et al., J. Biol. Chem. 270:494-502 (1995); Chen, K. et al., J. Biol. Chem. 273:749-755 (1998)), a signaling molecule that responds to unfolded protein in the ER to induce transcription of the ER protein chaperone genes has not been identified.
It would thus be desirable to provide a mammalian signaling molecule that responds to unfolded protein in the ER to induce transcription of the ER protein chaperone genes. It would also be desirable to identify and characterize the human gene product that is equivalent to Ire1p of S. cerevisiae and functions as a proximal sensor for the UPR in mammalian cells. It would further be desirable to provide a method for protecting cells from the stressful condition of unfolded protein in the ER.